Re: lipophilic dye fare red with narrow excitation peak -John's input

[Dagmar Adeline Brüggemann]

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Dear John,

could you please elaborate what you mean with to play with the settings a bit? I am currently trying to image far red dyes. Though the PMTs which are mounted should theoretically be able to visualize emission spectra up to 800 nm, it appears that the Quantum efficiency is greatly reduced ( at least 80%) already around 650 nm.   Is there anything what I can do without having to buy another type of PMT?


Any help would be greatly appreciated,


Dagmar Brüggemann, PhD
Professor Animal Science and Food Quality
Rhine-Waal University of Applied Sciences
Marie- Curie Str. 12
47533 Kleve






________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of John Oreopoulos [[hidden email]]
Sent: Monday, October 22, 2012 17:16
To: [hidden email]
Subject: Re: lipophilic dye fare red with narrow excitation peak

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Hi Thomas,

Since the lipophilic probes like DiI, DiO, and DiD usually absorb and emit light strongly compared to mCherry (as in your case), perhaps you could switch over to the longer wavelength probe DiR (no commercial interest):

https://products.invitrogen.com/ivgn/product/D12731?ICID=search-product

This probe has a much lower absorption at 647 nm, but I bet you'll still be able to get plenty of signal to form an image with the same laser line. The emission is further out in the red around 780 nm, so you'll need a different emission filter (your existing filter might work if it's a long-pass), but the bleedthrough into your mCherry channel should be much more reduced since the absorption at 561 nm is less than 5%.

You didn't mention what type of imaging system you're using. If it's a laser scanning confocal with a PMT, you might need to play with the settings a bit. If it's a camera-based system (like a spinning disk confocal), you should have no problems since most CCDs can still detect out in that wavelength range. Don't bother trying to look for the probe in the eyepieces, though!


Cheers,


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-10-22, at 10:46 AM, Horn Thomas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello all,
> we are currently using DiD to label a structure on a micro fluidics device. However, the cells that we need to visualize do contain mCherry. Problem is that the absorption/excitation curve of DiD has a long tail "on the left" so that 561 excites it very well. As the DiD fluorescence is very strong we do get a lot of Signal in the mCherry channel when exciting with a 561nm laser -  even if we use a small band pass on the lower emission spectrum of mCherry. The DiD signal is very strong versus a weak mCherry so that spectral unmixing  is problematic.
>
> To cut a long story short: can someone recommend a lipophilic   dye that is good for 647 excitation but is not excited by 561nm.
>
> Kind regards,
> Thomas
>
>
>
>
> Dr. Thomas Horn,
> The Single Cell Unit, U1.46
> Department of Biosystems Science and Engineering (D-BSSE)
> Swiss Federal Institute of Technology Zurich (ETH)
> Mattenstrasse 26
> CH 4048 Basel
> Switzerland
> Phone: [phone]
> mail: [hidden email]

[John Oreopoulos]

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Hey Dagmar,

What I meant by "changing the settings" was simply what you just said - I'm not sure if the PMT will have sufficient sensitivity out that range, so Thomas in his case might have to increase the gain, open the pinhole, average more frames, etc. - all the usual things that can be done to try and improve the image quality in a light starved situation on a laser scanning confocal microscope.

Perhaps there is someone out there on this listserver who can comment a bit more on the topic of imaging with an laser scanning confocal out into wavelengths around 750-800 nm. The technology has changed a bit since I last used a laser scanner, so I don't know what the latest news is on that.


John Oreopoulos
Research Assistant
Spectral Applied Research
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2012-10-22, at 11:46 AM, Dagmar Adeline Brüggemann wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear John,
>
> could you please elaborate what you mean with to play with the settings a bit? I am currently trying to image far red dyes. Though the PMTs which are mounted should theoretically be able to visualize emission spectra up to 800 nm, it appears that the Quantum efficiency is greatly reduced ( at least 80%) already around 650 nm.   Is there anything what I can do without having to buy another type of PMT?
>
>
> Any help would be greatly appreciated,
>
>
> Dagmar Brüggemann, PhD
> Professor Animal Science and Food Quality
> Rhine-Waal University of Applied Sciences
> Marie- Curie Str. 12
> 47533 Kleve
>
>
>
>
>
>
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on behalf of John Oreopoulos [[hidden email]]
> Sent: Monday, October 22, 2012 17:16
> To: [hidden email]
> Subject: Re: lipophilic dye fare red with narrow excitation peak
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Thomas,
>
> Since the lipophilic probes like DiI, DiO, and DiD usually absorb and emit light strongly compared to mCherry (as in your case), perhaps you could switch over to the longer wavelength probe DiR (no commercial interest):
>
> https://products.invitrogen.com/ivgn/product/D12731?ICID=search-product
>
> This probe has a much lower absorption at 647 nm, but I bet you'll still be able to get plenty of signal to form an image with the same laser line. The emission is further out in the red around 780 nm, so you'll need a different emission filter (your existing filter might work if it's a long-pass), but the bleedthrough into your mCherry channel should be much more reduced since the absorption at 561 nm is less than 5%.
>
> You didn't mention what type of imaging system you're using. If it's a laser scanning confocal with a PMT, you might need to play with the settings a bit. If it's a camera-based system (like a spinning disk confocal), you should have no problems since most CCDs can still detect out in that wavelength range. Don't bother trying to look for the probe in the eyepieces, though!
>
>
> Cheers,
>
>
> John Oreopoulos
> Research Assistant
> Spectral Applied Research
> Richmond Hill, Ontario
> Canada
> www.spectral.ca
>
>
> On 2012-10-22, at 10:46 AM, Horn Thomas wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hello all,
>> we are currently using DiD to label a structure on a micro fluidics device. However, the cells that we need to visualize do contain mCherry. Problem is that the absorption/excitation curve of DiD has a long tail "on the left" so that 561 excites it very well. As the DiD fluorescence is very strong we do get a lot of Signal in the mCherry channel when exciting with a 561nm laser -  even if we use a small band pass on the lower emission spectrum of mCherry. The DiD signal is very strong versus a weak mCherry so that spectral unmixing  is problematic.
>>
>> To cut a long story short: can someone recommend a lipophilic   dye that is good for 647 excitation but is not excited by 561nm.
>>
>> Kind regards,
>> Thomas
>>
>>
>>
>>
>> Dr. Thomas Horn,
>> The Single Cell Unit, U1.46
>> Department of Biosystems Science and Engineering (D-BSSE)
>> Swiss Federal Institute of Technology Zurich (ETH)
>> Mattenstrasse 26
>> CH 4048 Basel
>> Switzerland
>> Phone: [phone]
>> mail: [hidden email]